Dictyostelium has seven class-I myosins: three long-tailed (MIB, MIC, MID), three short-tailed (MIA, MIE, MIF), and one without a tail (MIK). The single heavy chains of these myosins have a motor domain, a neck and a non-helical tail. The tail of the long-tailed myosins contains a basic region with a basic-hydrophobic membrane-binding site (BH-site), a Gly-Pro-Gln (GPQ) region that binds F-actin, and a Src-homology 3 (SH3). The BH-site binds to acidic phospholipids. The GPQ region binds F-actin in both the presence and absence of ATP, unlike the ATP-sensitive actin-binding site in the motor domain. The SH3 region binds CARMIL, and perhaps other proteins. The short-tailed myosins contain the basic region, but not the GPQ region or SH3 domain. We showed previously that the cellular localization of long-tailed MIB is very dynamic, paralleling the rapidly changing cell morphology, and determined the regions of the heavy chain responsible for targeting MIB to different cell compartments. MIB localizes uniformly to the plasma membrane of freshly plated, non-motile cells. During random movement of the amoebae, MIB relocates to other structures such as pseudopods, actin waves (dynamic structures associated with the basal plasma membrane), endocytic protrusions, cell-cell contacts, and the front of polarized, chemotaxing cells. The BH site is required for MIB localization to the plasma membrane, both the BH site and the GPQ region are required for MIB localization to actin waves and macropinocytic protrusions, and the motor domain is both required and sufficient for localization of MIB at the front of polarized cells. The dynamic localizations of short-tailed MIA are consistent with the roles of the motor domain, BH-site and GPQ region in the localization of MIB. MIA localizes to the regions of the cell that require only the motor domain or the BH site for localization of MIB. And MIA does not localize to the regions where localization of long-tailed MIB requires the GPQ region, which short-tailed MIA does not have. Like MIA, the two other short-tailed myosin Is, MIE and MIF, localize uniformly to the plasma membrane, to cell-cell contacts, and diffusely in pseudopods and the front of chemotaxing cells. Unexpectedly, however, unlike short-tailed MIA, short-tailed MIE and MIF localize to macropinocytic cups, macropinocytic protrusions and actin waves. This year we continued the study on localization of short-tailed myosins MIE and MIF with special attention to actin waves and macropinocytic cups, i.e. the structures where localizations of myosin Is differ the most. At the beginning of macropinocytic cup formation long-tailed MIB and short-tailed MIE and MIF localize uniformly along the early protrusions. However, at later stages, i.e. during cup closure, MIB localizes only at cup edges whereas MIE and MIF are present on the whole cup surface. In actin waves, MIB localizes together with actin in actin waves whereas MIE and MIF are absent there and localize to the region encircled by the actin wave. Thus, in both actin waves and macropinocytic cups these three myosin Is form a striking bulls-eye structure with MIF and MIE in the middle and MIB at the periphery. Recently we found that, as for MIB and MIA, the BH region is required for localization of MIF to the plasma membrane. However, contrary to MIB and MIA, the BH region by itself is not sufficient. Plasma membrane localization of MIF also requires the neck domain, and a construct containing just the tail and neck region of MIF shows very strong affinity for macropinocytic structures.. We are currently characterizing the localizations of the other two long-tailed myosin Is, MIC and MID. We find that MID localizes to the region encircled by actin waves, similar to the localization of short-tailed MIE and MIF and different than the localization of long-tailed MIB. These similarities and differences can be explained by the high affinity of MID, MIE and MIF for PI(3,4,5)P3, which had been shown to be at the center of actin waves. Preliminary data show that the localization of MID in macropinocytic cups also resembles the localizations of MIF and MIE (and not MIB), i.e. along the whole cup surface and not enriched at cup edges. Finally, and surprisingly, long-tailed MIC shows strong association with filopodia and cell protrusions that we have not observed for the other myosin Is. Summarizing, our earlier and present data show that the Dictyostelium myosin Is have overlapping as well as unique localizations, the biggest differences occurring in macropinocytic cups and actin waves. The role of actin waves is not clear but differential localization of myosin I in macropinocytic cups seem to be of particular importance since, for example, myosin Is have been shown to play a role in macropinocytosis of Rac-activated cancers in mammals.